11/14/2006
– HCV Therapy – Pre-Clinical and Early Clinical Development
Abstract ID: 67405
Category: JO6: HCV Therapy: Preclinical and Early Clinical Development
Therapeutic vaccination for chronic hepatitis C virus infection in HCV trimera mice.
F.
Rahman, I. Department of Internal Medicine, University Mainz, Germany, Mainz,
Germany,
N. Blust, I. Department of Internal Medicine, University Mainz, Germany,
Mainz,
Germany, T. Baumert, Internal Medicine II,, Freiburg, Germany, M. Geissler,
Internal
Medicine II,, Freiburg, Germany, P. R. Galle, I. Department of Internal
Medicine,
University Mainz, Germany, Mainz, Germany, W. O. Böcher, I. Department
of
Internal Medicine, University Mainz, Germany,
Discussion
Whereas spontaneous resolution from hepatitis C is
associated with strong antiviral T cell responses in the acute phase of the
disease, only weak immune responses are found in patients developing chronic
infection. Thus, induction of strong HCV-specific T cell responses could
represent a new therapeutic strategy. Virus like particles (VLP) expressing the
HCV structural proteins as well as synthetic peptides representing well
characterized CTL epitopes might serve as an effective therapeutic vaccine. The
humanized trimera mouse model was used to study the immunogenicity of these
vaccine candidates in a preclinical animal model. Irradiated Balb/c mice were
transplanted with nod.scid mouse bone marrow and reconstituted with human blood
lymphocytes (PBMC) from three HLA A2.1 positive chronic HCV patients. These
so-called trimera mice were vaccinated intraperitoneally with VLP expressing
the structural proteins core, E1 and E2 or with a mixture of HLA A2 restricted
synthetic peptide CTL epitopes from the HCV core, E1, E2 or NS3 region. PBMC
were recovered 10 days after vaccination and T cell analysis was performed with
tetramer staining or gIFN Elispot assay with recombinant HCV antigens,
overlapping HCV peptides or the respective CTL epitopes. HCV specific T cell
responses could be detected in trimera mice reconstituted with PBMC from two
out
of three chronic HCV patients, although such responses
were undetectable in donor PBMC. Alternatively, instead of PBMC reconstitution,
HCV-infected human liver tissue was implanted under the kidney capsule of the
trimera mice. HCV quantification in the trimera serum and HE-staining of implanted
liver grafts was performed at different time points. Viremia was detectable in
several mice for more than three weeks after implantation and histological
staining showed viable liver tissue up to this point. Since the trimera mouse
model supports both, induction of viremia and immune responses, it should allow
preclinical testing of new vaccine candidates, such as lipopeptides,
exosomes or dendritic cells, making a therapeutic
vaccination a potentially successful approach for the future.
Abstract ID: 64638
Category: JO6: HCV Therapy: Preclinical and Early Clinical Development
Demonstration of the Efficacy of Small Molecule HCV Inhibitors in the KMT MouseModel of Hepatitis C Virus (HCV) Infection.
N.
Kneteman, University of Alberta Hospital,
Aim
We report efficacy of clinically proven inhibitors of
HCV infection (Interferon alpha 2b, BILN 2061) in our mouse model, thus
demonstrating the utility of the model for HCV antiviral drug discovery.
MATERIALS
AND METHODS
SCID carrying the urokinase type plasminogen activator
gene (KMT mice) were transplanted with human hepatocytes to yield mice with
chimeric human livers (Nature Medicine 7:927, 2001). Mice with HCV
titres104-107 copies/ml received 1350 IU/gram body INFalpha,(n=18 X 2 wk; n=4 X
4 wk) or saline (n=16 X 2 wk; n=4 X 4 wk) by IM daily. Nine mice received
10mg/kg bid orally of the protease inhibitor BILN2061for 4 (n=3) or 7(n=6)
days. Mice with increasing titre (after initial drop) despite ongoing treatment
with BILN2061 had RT-PCR products from plasma/liver sequenced to evaluate
development of viral escape mutations as previously reported in the replicon
system during BILN 2061 administration.
RESULTS
INF resulted in a consistent decrease in viral titre
of 0.7, 1.1, and 2.0 log after 1, 2 and 4 wk (P<0.01 at all time points vs
control). Therapy with BILN2061 resulted in viral titres dropping mean 2 log
after 4 and 1.2 log after 7 days (P<0.0001 BILN2061 vs. vehicle, P<0.03
/BILN vs. INF). The D168V mutation previously reported to be associated with
resistance to BILN2061 in replicon cultures (J. Virol. (2003) 77:3669) was not
found in the sera or liver. Pre-existing mutants (Q80K) with partial resistance
to
BILN2061 were identified.
CONCLUSIONS
Results with INF and BILN2061 therapy of HCV infection
in the KMT mouse model of HCV infection parallel reported outcomes in clinical
application (Nature 426:186, 2003), validating the KMT model for evaluation of
potential anti-HCV therapeutics. HCV escape mutations previously reported
during BILN2061 treatment of HCV replicons in vitro were not seen in this in
vivo system. This system should help advance into human trials, lead compounds
with an increased likelihood of clinical success while broadening the tools
available for study of the biology of HCV infection.
Abstract ID: 65035
Category: JO6: HCV Therapy: Preclinical and Early Clinical Development
Pharmacokinetics, Safety, and Tolerability of the Isatoribine Oral Prodrug ANA975 in aPhase 1 Healthy Volunteer Study.
B.
Kerr, Anadys Pharmaceuticals Inc, San Diego, CA, L. Bauman, Anadys
Pharmaceuticals,
Inc., San Diego, CA, S. Webber, Anadys Pharmaceuticals, Inc., San
Diego,
CA, A. Xiang, Anadys Pharmaceuticals, Inc., San Diego, CA, J. Ng, Anadys
Pharmaceuticals,
Inc., San Diego, CA, L. Kirkovsky, Anadys Pharmaceuticals, Inc., San
Diego,
CA, D. Bartkowski, Anadys Pharmaceuticals, Inc., San Diego, CA, K. Steffy,
Anadys
Pharmaceuticals, Inc., San Diego, CA, S. Fletcher, Anadys Pharmaceuticals,
Inc.,
San Diego, CA, R. Aust, Anadys Pharmaceuticals, Inc., San Diego, CA, J. Theiss,
SunCoast
Tox, San Diego, CA, D. Averett, Anadys Pharmaceuticals Inc,
PURPOSE
In a proof-of-concept (POC) clinical study,
intravenous (IV) infusion of the
TLR7 agonist isatoribine 800mg once daily x 7 days to
patients chronically infected with hepatitis C virus (HCV) yielded a
significant reduction of plasma HCV RNA that correlated with induction of 2’,
5’-oligoadenylate synthetase (OAS). Oral doses of isatoribine are poorly
absorbed in multiple species. The prodrug ANA975 was developed to improve oral
delivery of isatoribine to systemic circulation.
METHODS
Toxicology studies, including GLP oral multiple-dose
studies in cynomolgus monkeys, were performed to enable clinical studies. An
open label, rising
dose level, single-dose study in healthy volunteers
was performed in the
RESULTS
Oral administration of ANA975 to monkeys resulted in
efficient systemic
delivery of isatoribine; the highest tolerated dose in
monkey achieved plasma area-underthe-curve (AUC) values 6-13x higher than achieved
with the 800mg IV dose in the clinical POC study. Monkeys showed a dose-related
OAS induction in blood that persisted with once daily dosing for treatment
periods up to at least 28 days. Single oral doses of ANA975 were well tolerated
by healthy volunteers. There were no serious adverse events (AEs) and no
withdrawals from the study. A total of 15 AEs were reported by 13 of 36
subjects. All reported AEs were mild (13 events) or moderate (2
events) in severity. The Investigator considered 1 AE
(mild transient increase in CPK at 800mg) to be possibly related to ANA975 and
all other AEs to be unlikely related or not related to ANA975. There were no
reports of gastrointestinal AEs or flu-like syndrome. ANA975 was rapidly and
extensively converted to isatoribine, with isatoribine Cmax typically occurring
within 1 hour post-dose. At the highest tested dose, which has an
isatoribine dose content of 988mg, plasma isatoribine
AUC values (range 23-40, median 27mg*h/L) were similar to those observed in the
clinical POC study with daily 1-hour IV infusions of isatoribine 800mg (first
dose AUC range 19-38, median 24mg*h/L). The similarity of AUC values at
comparable molar doses of oral ANA975 and IV isatoribine indicates that
fractional oral absorption and conversion of ANA975 to isatoribine was very
high.
CONCLUSION
ANA975 is an efficient oral prodrug that achieves
plasma isatoribine exposures comparable to those previously shown to reduce
plasma HCV RNA in chronically infected patients. The data is encouraging and support further
studies of ANA975.
Abstract ID: 60761
Category: JO6: HCV Therapy: Preclinical and Early Clinical Development
Decline in serum neopterin concentration correlates with HCV RNA decline during administration of VX-950, a Hepatitis C Virus Protease Inhibitor.
H. C.
Gelderblom, Academic Medical Center, Department of Gastroenterology and Hepatology,
Amsterdam, Netherlands, S. Zeuzem, Saarland University Hospital, Homburg/Saar,
Germany, C. J. Weegink, Academic Medical Center, Department of Gastroenterology
and Hepatology, Amsterdam, Netherlands, N. Forestier, Saarland University
Hospital, Homburg/Saar, Germany, L. McNair, Vertex Pharmaceuticals, Cambridge,
MA, S. Purdy, Vertex Pharmaceuticals, Cambridge, MA, P. L. Jansen, Academic Medical Center, Department of Gastroenterology and
Hepatology, amsterdam, Netherlands, H. W. Reesink, Academic Medical Center,
Department of Gastroenterology and Hepatology,
Background
Neopterin is a guanosine triphosphate (GTP) derived
compound that is produced by activated monocytes/macrophages. We followed
neopterin levels during administration of VX-950, an orally administered
inhibitor of the Hepatitis C virus (HCV) NS3·4A protease, in a multiple-dose
study in 34 patients chronically infected with HCV genotype 1.
Methods
VX-950 was administered for 14 days at doses of 450 mg
or 750 mg every 8
hours, or 1250 mg every 12 hours, or placebo. Serum
neopterin levels were measured by immunoassay at days -1, 7 and 14 of dosing,
and day 10 of follow-up (day 24). HCV RNA was assessed by real-time PCR.
Results
In patients with chronic HCV infection, VX-950 had
substantial antiviral
effects, with every patient demonstrating at least a
2-log drop in viral load in all dosing groups. In the 750 mg q8h dose group, there
was a reduction in median HCV RNA of more than 3 log10 after 3 days, and of 4.4
log10 at the end of 14 days of dosing. In the 450 mg q8h and 1250 mg q12h dose
groups, maximal effect was seen between 3 and 7 days of dosing followed by an
increase in median viral load between days 7 and 14. Median viral loads
increased in all groups between days 14-24 (post-dosing). Baseline neopterin
levels were elevated in 23/34 patients (median 9.45
nmol/l; ULN 7.7 nmol/l). In the 750 mg q8h dose group, the changes from
baseline in median neopterin level were -3.6, -3.8 and -1.3 nmol/l at days 7,
14 and 24 (figure). In the 450 q8h dose group, the changes were -1.8, -1.4 and
+0.4 nmol/l at days 7, 14 and 24 (figure). In the 1250 q12h dose group, the
changes were, +0.3, -0.2 and + 1.0 nmol/l at days 7, 14 and 24 (figure). In the
placebo group, the changes from baseline in median neopterin level were -0.2,
+0.5, and +0.4 nmol/l at days 7, 14 and 24. Median ALT levels, which were
elevated at baseline, normalized during 14 days of dosing in all groups.
Discussion
Changes in median neopterin levels correlated with the
decrease in HCV
RNA and ALT during administration of VX-950. The
maximal decrease in median neopterin level was in the 750 mg q8h dose group,
which was also the dose group with maximal reductions in HCV RNA. These data
suggest that inhibition of HCV replication by VX-950 abrogates inflammation.
Abstract ID: 66459
Category: JO6: HCV Therapy: Preclinical and Early Clinical Development
Characterization of NM283 (Valopicitabine) Resistance Profile Using Bovine Viral Diarrhea Virus.
V.
Bichko, Idenix Pharmaceuticals, Inc., Cambridge, MA, L. Qu, Idenix
Pharmaceuticals, Inc., Cambridge, MA, M. La Colla,
Idenix Pharmaceuticals, Inc.,
Cambridge,
MA, M. Tausek, Idenix Pharmaceuticals, Inc., Cambridge, MA, S.
Bergelson,
Idenix Pharmaceuticals, Inc., Cambridge, MA, C. Pierra, Laboratoire
Coopératif
Idenix-CNRS-Université Montpellier II, Montpellier, R. Storer, Idenix
Pharmaceuticals,
Inc., Cambridge, MA, G. Gosselin, Laboratoire Coopératif Idenix-
CNRS-Université
Montpellier II, Montpellier, J. Sommadossi, Idenix Pharmaceuticals,
Inc.,
Cambridge, MA, D. Standring, Idenix Pharmaceuticals, Inc.,
Background
Valopicitabine (NM283) reduces serum hepatitis C virus
(HCV) RNA
levels in patients and in chronically infected
chimpanzees, and is currently in phase II clinical development for the
treatment of chronic hepatitis C. NM283 is an orally bioavailable prodrug of
NM107 (2’-C-methylcytosine), which inhibits flavi- and pestivirus replication
in vitro and shows enhanced antiviral activity in combination with interferon
alfa. The present study evaluated the potential for the development of
resistance to NM283 using NM107 and a bovine viral diarrhea virus (BVDV) in vitro
infection model. BVDV is a pestivirus related to HCV.
Methods
NM107-resistant BVDV variants were isolated by
passaging infected DBK
cells with varying concentrations of NM107. Both cytopathic
(cp) and noncytopathic (ncp) BVDV biotypes were evaluated. Resistant mutants
were characterized in terms of replication fitness (see below) and sensitivity
to NM107 and Intron A. A mutation within the NS5B region was confirmed by
sequencing.
Results
Repeated passaging of a cp strain of BVDV (NADL) with
NM107 failed to
produce resistant mutants. However, although prolonged
NM107 treatment can eradicate BVDV in a cell line persistently infected with
ncp BVDV, lower concentrations of NM107 led to selection of resistant virus
after 3 to 5 cell passages. Sequencing revealed resistance was due to a S405T
amino acid substitution near the start of the B domain motif of the BVDV NS5B
polymerase, confirming that the polymerase is the target for NM107. This motif
is highly conserved among positive-strand RNA viruses, including
HCV, predicting that the equivalent mutation in HCV
polymerase is S282T. The NM107- resistant BVDV strains show at least 50-fold
reduced susceptibility to NM107, accompanied by decreased replication fitness
in vitro: “small plaque” phenotype, slow growth kinetics and lower virus
titers. Importantly, the S405T mutant virus showed ~38 fold increased
susceptibility to IFN a-2b compared to wild type BVDV. Consistent with
this finding, no viral resistance developed in vitro
when NM107 was used in combination with Intron A. To date, we have been unable
to find additional compensatory mutations that restore the phenotype of the
S405T mutant BVDV to that of wild type.
Conclusions
Resistance of BVDV to NM107 (due to the S405T mutation
in NS5B) was associated with reduced replication fitness, a lack of
compensatory mutations, and increased
sensitivity to Intron A. The homologous mutation in HCV NS5B is S282T. However,
this mutant (or NM283 resistance) has yet to be observed in the clinic after up
to 6 months therapy with interferon alfa 2b and NM283 in the ongoing phase II
clinical trial.
Abstract ID: 66647
Category: JO6: HCV Therapy: Preclinical and Early Clinical Development
REDIRECTING T LYMPHOCYTE SPECIFICITY USING HCV-SPECIFIC T CELL RECEPTOR GENES CONFERS RECOGNITION OF NATURALLY OCCURRINGVIRAL MUTANTS.
H. R.
Rosen, PVAMC, Portland, OR, G. Callender, U. Chicago, Chicago, IL, G. E.
Lyons,
University of Chicago, Chicago, IL, M. Nishimura, U. Chicago,
Background: HCV infection is manifested by a high rate
of viral persistence related to high viral mutability as well as diminished CTL
effector function. We have previously identified a T cell receptor (TCR) that
has relatively high affinity for HLA A2-restricted HCV NS3 1406-1415 peptide.
The purpose of this study was to determine whether gene transfer of this TCR
into peripheral blood lymphocytes (PBL) from normal healthy controls could
induce HCV-specific reactivity that would respond to wild-type and
mutant virus.
Methods
TCR alphaƒnand beta chain genes from the HCV-specific
TCR were cloned
and inserted into a retroviral vector capable of
delivering the genes into human T cells. The ability of transferred TCR genes
to confer HCV reactivity to PBL from 3 healthy donors was evaluated using
IFN-gamma production following stimulation with wild type or mutant peptide.
Results
TAP-deficient T2 cells were loaded with wild type NS3
1406-1415 peptide
(KLVALGINAV), eight different naturally occurring
viral mutants (V1408L, A1409T, I1412V, I1412L, I1412N, V1408T and the multiple
substituted peptides A1409G/I1412L/V1408T and
1408S/A1409G/I1412L), or a melanoma control peptide. TCR-transduced PBL
(both CD4+ and CD8+ T cells) produced statistically significant amounts of
IFN-gamma following stimulation with wild type HCV peptide and 7 of the 8 HCV
mutant peptides but not with melanoma peptide. Moreover, TCR-transduced PBL
recognized endogenously processed peptide.
Conclusions
We demonstrate the ability to engineer normal
PBL-derived T cells to
specifically recognize HCV independent of immune status. By providing T cell help as well as effector function, this approach represents a promising immunotherapy. Moreover, the ability of TCR-transduced PBL to recognize mutants suggests this approach might prevent antigenic escape variants.
Abstract ID: 66787
Category: JO6: HCV Therapy: Preclinical and Early Clinical Development
Single Dose Pharmacokinetics of a Novel Hepatitis C Protease Inhibitor, SCH 503034, in an Oral Capsule Formulation.
J.
Zhang, Schering-Plough Research Instutute, Kenilworth, NJ, S. Gupta, Schering-
Plough
reserach Institute, kenilworth, NJ, R. Rouzier, Centre Cap, Montpelleir,
France,
A.
Calzetta, Schering-Plough Research Institute, Kenilworth, NJ, D. L. Cutler,
Schering-Plough
Research Institute,
Background
SCH 503034, is an orally active Hepatitis C virus NS3
serine protease inhibitor that exhibits potent and specific in vitro antiviral
activity. We report results from our Phase I clinical development program
evaluating the pharmacokinetics (PK) and safety of SCH 503034 oral capsules in
healthy subjects.
Methods
In this randomized, single rising dose, double-blind
study, healthy subjects were randomized 2:1 to receive, SCH 503034 oral
capsules, (n=6/group ) 50 mg, 100 mg, 200 mg, 400 mg, 600 mg, 800 mg or placebo
(n=3/group). Plasma samples were collected at multiple time points to 120h
post-dose. Samples were analyzed by validated LC-MS/MS with a LOQ of 0.5 ng/ml.
Pharmacokinetics were analyzed using model independent methods. Safety was
assessed by clinical laboratory tests, vital signs, ECGs, physical exams and
occurrence of adverse events.
Results
Fifty-four subjects completed the study, 36 received
SCH 503034, 18 received placebo. SCH 503034 was rapidly absorbed following oral
administration of capsules (mean Tmax :1-2.25 h across the 6
dosing levels). After attaining Tmax, plasma SCH503034
concentrations declined in a bi-phasic manner, with a mean terminal phase
half-life (T1/2 ) of 7.0 to 15 h. Cmax and AUC increased in a dose-related
manner. The safety profiles were similar in subjects receiving SCH 503034 and
placebo.
Conclusions
SCH 503034 was readily bioavailable when administered
as a single dose in an oral capsule formulation and was well tolerated in
subjects receiving up to 800 mg orally.
Abstract ID: 59834
Category: JO6: HCV Therapy: Preclinical and Early Clinical
Development Identification of a Novel Small Molecule Hepatitis C Virus Replication Inhibitor that Targets Host Sphingolipid Biosynthesis.
H.
Sakamoto, Kamakura Research Laboratories, Chugai Pharmaceutical Co. Ltd.,
Kamakura,
Japan, K. Okamoto, Kamakura Research Laboratories, Chugai
Pharmaceutical
Co. Ltd., kamakura, Japan, M. Aoki, Kamakura Research Laboratories,
Chugai
Pharmaceutical Co. Ltd., Kamkura, Japan, H. Kato, Kamakura Research
Laboratories,
Chugai Pharmaceutical Co. Ltd., Kamakura, Japan, A. Katsume,
Kamakura
Research Laboratories, Chugai Pharmaceutical Co. Ltd., Kamakura, A. Ohta,
Kamakura
Research Laboratories, Chugai Pharmaceutical Co. Ltd., Kamakura, Japan,
T.Tsukuda, Kamakura Research Laboratories, Chugai Pharmaceutical Co. Ltd.,
Kamakura, Japan, N. Shimma, Kamakura Research Laboratories, Chugai
Pharmaceutical Co. Ltd.,Kamakura, Japan, Y. Aoki, Kamakura Research
Laboratories, Chugai Pharmaceutical Co. Ltd., Kamakura, Japan, M. Arisawa,
Kamakura Research Laboratories, Chugai Pharmaceutical Co. Ltd., Kamakura,
Japan, M. Kohara, Tokyo Metropolitan Institute of Medical Science, Tokyo,
Japan, M. Sudoh, Kamakura Research Laboratories, Chugai Pharmaceutical Co.
Ltd.,
Introduction
An estimated 170 million individuals worldwide are
infected with hepatitis C virus (HCV), a serious cause of chronic liver
disease. Current interferon-based therapy for treating HCV infection has an
unsatisfactory cure rate, and the development of more efficient drugs is
needed. Here, we have identified a lipophilic long-chain base compound, NA255,
from a secondary fungal metabolite, as a novel small molecule HCV replication
inhibitor using an HCV subgenomic replicon cell culture system. HCV replicon 1b
cell-based assay showed that the replication was suppressed by NA255 in a
dose-dependent manner with a mean of 50% inhibitory concentration (IC50) of 2
nM. NA255 had no effect on host-cell viability (IC50>50,000 nM) and cell
cycle progression. We found that NA255 prevents the de novo synthesis of
sphingolipids, major lipid raft components, thereby inhibiting serine
palmitoyltransferase and disrupting the association among HCV nonstructural
(NS) viral proteins on the lipid rafts. To determine whether
HCV protein could interact directly with
sphingolipids, we searched for the sphingolipidbinding domain (SBD) in HCV NS
protein. We found that NS5B protein has a SBD in the molecular structure and
that the interaction of NS5B with sphingolipids could be involved in HCV
replicon replication.
Conculsion
Thus, NA255 is a potent anti-HCV replication inhibitor
that targets host lipid raft, suggesting that inhibition of sphingolipid
metabolism may provide a novel therapeutic strategy for treatment of HCV
infection.
Abstract ID: 60227
Category: JO6: HCV Therapy: Preclinical and Early Clinical
Development SCH 503034, a Mechanism-based Inhibitor of Hepatitis C Virus (HCV) NS3 Protease Suppresses Polyprotein Maturation and Enhances the Antiviral Activity of Interferon-á-2b (INF)
B. A.
Malcolm, Schering-Plough Research Instutute, Kenilworth, NJ, A. Arassappan,
Schering
Plough Research Institute, Kenilworth, NJ, F. Bennett, Schering-Plough
research
Institute, Kenilworth, NJ, S. Bogen, Schering-Plough research Institute,
Kenilworth,
NJ, R. Chase, Schering-Plough research Institute, Kenilworth, NJ, K. Chen,
Schering-Plough
research Institute, Kenilworth, NJ, T. Chen, Schering-Plough research
Institute,
Kenilworth, NJ, P. Ingravallo, Schering-Plough research Institute, Kenilworth,
NJ,
E. Jao, Schering-Plough research Institute, Kenilworth, NJ, S. Kong, Schering-
Plough
research Institute, Kenilworth, NJ, F. Lahser, Schering-Plough research
Institute,
Kenilworth,
NJ, R. Liu, Schering-Plough research Institute, Kenilworth, NJ, Y. Liu,
Schering-Plough
research Institute, Kenilworth, NJ, R. Lovey, Schering-Plough research
Institute,
Kenilworth, NJ, J. McCormick, Schering-Plough research Institute,
Kenilworth,
NJ, G. F. Njoroge, Schering-Plough research Institute, Kenilworth, NJ, A.
Saksana,
Schering-Plough research Institute, Kenilworth, NJ, A. Skelton, Schering-
Plough
research Institute, Kenilworth, NJ, X. Tong, Schering-Plough research
Institute,
Kenilworth,
NJ, S. Venkatraman, Schering-Plough research Institute, Kenilworth, NJ, J.
Wright-Minogue,
Schering-Plough research Institute, Kenilworth, NJ, E. Xia, Schering-
Plough
research Institute, Kenilworth, NJ, V. Girijavallabhan, Schering-Plough
research
Institute,
Background
Cleavage of the HCV polyprotein by the NS3 protease
releases proteins essential for viral propagation. Additionally, NS3-mediated
cleavage of host factors integral to Interferon Response Factor-3 signaling may
result in abrogated cellular responses to IFN. Thus, blockage of NS3 activity
is expected to inhibit HCV replication by direct suppression of viral
production and restoration of host IFN responsiveness. SCH 503034, reversibly
binds the HCV NS3 protease and specifically inhibits its activity.
Methods
SCH 503034 NS3 binding activity was determined
spectrophotometrically. Anti-viral potency was assayed in HCV replicon cells
exposed to increasing amounts of SCH 503034 for various times up to 15d. HCV
replicon RNA and protein expression were monitored by Northern and Western
analysis, respectively and replicon copy number was determined by qRT-PCR
multiplex reaction. The effect of SCH 503034 on
response to IFN was evaluated in replicon cells incubated with increasing
amounts of SCH 503034 and IFN for 72h. Toxicity, was assessed by cell viability
and doubling time.
Results
SCH 503034 exhibited time-dependent inhibition of
single chain NS3 with a binding constant (Ki*) equal to ~14 nM. SCH 503034
inhibition of replicon synthesis was dose-related. The IC50 and IC90 for
suppression of replicon synthesis following 72-h exposure were ~200 nM and 400
nM, respectively. Exposure of replicon cells to SCH 503034 over 15d resulted in
log
reductions in replicon RNA of ~ 2.0, 3.5, and 4.0 at
1.2, 6 and 12 x IC90, respectively. Isobologram analysis revealed that SCH
503034 + IFN-
α was more effective than the line of additivity,
though the difference was not significant (p>0.1). No toxicity was seen
after exposure of HuH-7 cells to 50μM SCH 503034 for 96 h, or primary
hepatocytes to 10 x IC90 for 1 wk. No changes in doubling times or growth rates
of HuH-7 cells were seen after a 3-wk at 10 x IC90.
Conclusions
SCH 503034 exhibited potent anti-viral activity both
alone and in combination with IFN-α in the HCV replicon assay. The
combination of SCH 503034 with IFN may lead to enhanced clinical efficacy.
Abstract ID: 67060
Category: JO6: HCV Therapy: Preclinical and Early Clinical Development
HCV replication is inhibited by cyclosporin A, mycophenolic acid and IFN-alpha in a synergistic fashion and with distinct anti-viral kinetics.
S. Henry, Erasmus MC, Rotterdam, Netherlands, H.
Metselaar, Erasmus MC,
Rotterdam, Netherlands, R. Bartenschlager,
Otto-Meyerhof Center, Heidelberg,
Germany, H. Tilanus, Erasmus MC, Rotterdam,
Netherlands, L. van der Laan, Erasmus
MC,
Background
Chronic Hepatitis C Virus (HCV) infection is the
leading cause of liver transplantation worldwide. The success of
transplantation, however, is compromised by the re-infection of the graft and
this problem has begun to worsen in the last decade. Changes in
immunosuppressive therapy may have
contributed to this deterioration. The aim of this
study is to determine the effect of different immunosuppressive compounds on
HCV replication and determine the synergy and kinetics of inhibition.
Methods
The anti-viral properties of pegylated IFN-alpha 2b
(IFN) and immunosuppressive drugs were tested in vitro using an HCV-replication
model. Huh-7 hepatoma cells, containing the HCV replicon with a
luciferase reporter gene, were cultured for 18 hour
with different doses or combinations of drugs. HCV replication was quantified
based on luciferase activity, RT-PCR for viral RNA and immunocytochemistry for
helicase (NS3) protein. Kinetics of inhibition was determined with the Xenogen
IVIS imaging system.
Results
Tacrolimus, rapamycin, dexamethason or prednisolon did
not inhibit HCV replication, whereas almost complete inhibition (98%) was
observed with IFN (10IU/ml). Both cyclosporin A (CsA) (0.5-5.0 ug/ml) and
mycophenolic acid (MPA) (2.0-6.0 ug/ml) blocked HCV replication up to 70%. No
cell death was observed with any tested compounds. Proliferation of Huh-7 cells
was reduced with MPA, but did not account for the observed inhibition of HCV
replication. When CsA and MPA were combined, synergistic inhibition was observed
up to 90% at higher doses. Synergistic inhibition was also observed with a
combination of CsA and suboptimal doses of IFN. The anti-viral kinetics of CsA,
MPA and IFN were shown to be different (See Fig).
Conclusion
The immunosuppressive drugs CsA and MPA both are
potent and specific inhibitors of HCV replication. The synergy and different
anti-viral kinetics suggest that antiviral activity of both drugs act via
independent pathways and can operate independent of each other. These findings
suggest that
immunosuppressive therapy based on a combination of
CsA and MPA (low or free of steroids) is preferable for reducing HCV recurrence
after transplantation.
Abstract ID: 61330
Category: JO6: HCV Therapy: Preclinical and Early Clinical Development
NIM811 exhibits potent anti-HCV activity in vitro and represents a novel approach for viral hepatitis C therapy.
K.
Lin, Novartis Institutes of Biomedical Research, Cambridge , MA, S. Ma,
Novartis
Institutes
of Biomedical Research, Cambridge, MA, J. Boerner, Novartis Institutes of
Biomedical
Research, Cambridge, MA, M. Huang, Novartis Institutes of Biomedical
Research,
Cambridge, MA, N. Ryder, Novartis Institutes of Biomedical Research,
Cambridge,
MA, B. Weidmann, Novartis Institutes of Biomedical Research, Cambridge,
MA,
M. P. Cooreman, Novartis,
Introduction/ Discussion
Host factors required in the lifecycle of hepatitis C virus (HCV) are potential targets of
antiviral therapy, and are complementary to inhibitors of viral enzymes such as NS3-4A
protease and NS5B polymerase. The anti-HCV effect of CsA has been demonstrated both
in vitro and in a controlled clinical trial. Various CsA analogs were evaluated for their
inhibitory effect on viral RNA replication in HCV replicon cells. There was a good
correlation between antiviral activity and cyclophilin-binding activity of these
compounds. NIM811 was selected for further research because it binds to cyclophilins
with similar affinity as CsA but is devoid of significant immunosuppressive activity, as
the NIM811-cyclophilin complex does not bind to calcineurin. NIM811 induced a
concentration-dependent reduction of HCV RNA in replicon cells with an IC50 of 0.66
ìM at 48 hours, compared to 0.80 ìM for CsA. After a nine-day treatment of replicon
cells, a three-log10 reduction of HCV RNA levels was achieved with as low as 1 ìM
NIM811. Moreover, NIM811 showed equally potent activity in HCV replicons that were
highly resistant to viral protease or polymerase inhibitors. Resistance against this
compound appears to be difficult to generate in vitro. In vitro, combinations of NIM811
with other HCV inhibitors produced at least additive antiviral effects. Pharmacokinetic
studies in rats showed a t1/2 of circa 14 hours, with hepatic concentrations 10-fold over
serum levels. Toxicology studies showed no safety issue at the dose range predictive of
therapeutic efficacy. In conclusion, NIM811 has potent anti-HCV activity and is several
orders of magnitude less immunosuppressive than CsA in vitro. The fact that it targets a
host protein and the demonstration of its uncompromised efficacy in HCV resistant
against protease or polymerase inhibitors provide the rationale for combining of NIM811
with other specific antivirals to improve therapeutic efficacy and to suppress emergence
of drug-escape mutants.
Abstract ID: 62612
Category: JO6: HCV Therapy: Preclinical and Early Clinical Development Genetic heterogeneity in the HCV NS3 protease of untreated genotype 1 patients has little effect on the sensitivity to VX-950.
T. Kieffer, Vertex Pharmaceuticals Inc.,
Cambridge, MA, C. Sarrazin,
Universitätsklinikum des Saarlandes, Homburg,
Germany, D. Bartels, Vertex
Pharmaceuticals Inc., Coralville, IA, B. Hanzelka,
Vertex Pharmaceuticals Inc.,
Coralville, IA, U. Muh, Vertex Pharmaceuticals
Inc, Coralville, IA, M. Welker,
Universitätsklinikum des Saarlandes, Homburg,
Germany, D. Wincheringer,
Universitätsklinikum des Saarlandes, Homburg,
Germany, C. Lin, Vertex
Pharmaceuticals
Inc., Cambridge, MA, T. Grossman, Vertex Pharmaceuticals Inc.,
Cambridge,
MA, C. Weegink, Academic Medical Center (AMC), Department of
Gasteroenterology
and Hep, Amsterdam, Netherlands, S. Purdy, Vertex Pharmaceuticals
Inc,
Cambridge, MA, H. Reesink, Academic Medical Center (AMC), Department of
Gasteroenterology and Hep, Amsterdam, Netherlands,
A. Kwong, Vertex
Pharmaceuticals Inc, Cambridge, MA, S. Zeuzem,
Universitätsklinikum des Saarlandes,
Introduction/Discussion
The non-structural (NS)3-4A protease is essential for
hepatitis C virus (HCV) replication and a promising target for new anti-HCV
therapy. VX-950, a potent and specific NS3-4A protease inhibitor, has recently
demonstrated significant antiviral activity in a phase 1b trial of patients
infected with HCV genotype 1 . Median reductions of 2 to 4 log10 IU
per mL in serum HCV RNA concentrations were achieved
after 14 days of dosing with VX-950. The degree to which a patient responds to
treatment and the rate at which viral rebound is observed could in part be due
to genotypic differences in sensitivity to the protease inhibitor. The rapid
replication rate of hepatitis C virus, along with the poor fidelity of its
polymerase, gives rise to an accumulation of mutations throughout its
genome. The degree to which sequence variability in
the protease region affects the catalytic efficiency of the enzyme or the
binding of an inhibitor is not known. Here, we characterize the extent of
sequence diversity within the NS3 protease domain of HCV isolated from 34
patients enrolled in the phase 1b trial, before dosing with VX-950. The NS3
protease domain was amplified by nested RT-PCR, cloned, and sequenced. The
consensus sequence for each patient’s HCV population was derived from an average
of
90 independent cDNA clones. The average intra-patient
amino acid quasispecies complexity (Shannon entropy) and diversity (Hamming
distance) was low (0.332 ± 0.109 and 0.421 ± 0.195, respectively) and no
correlation of quasispecies heterogeneity with HCV RNA serum concentration at
baseline was observed. The inter-patient (individual
consensus compared to genotype 1a or 1b consensus
sequence) amino acid diversity was 1% for genotype 1a and 2% for genotype 1b.
Modeling analysis predicted that amino acid differences observed between
consensus sequences of all these patients were expected to have little or no
impact on VX-950 binding. Patient specific protease clones were then expressed
and tested for inhibition by the protease inhibitor VX-950. In agreement with
the modeling observation, there were no significant differences in the
enzymatic IC50 of these proteases derived from
different patient isolates.
Conclusion
It appears that despite the observed sequence diversity
in the HCV NS3 serine protease, patients may be uniformly responsive to
treatment with the protease inhibitor VX-950.
Abstract ID: 67450
Category: JO6: HCV Therapy: Preclinical and Early Clinical Development
CANNABINOIDS BLOCK INTERFERON-MEDIATED SUPPRESSION OF HEPATITIS C VIRUS (HCV) REPLICATION.
S.
Ramesh, Virginia Commonwealth University, Richmond, VA, F. Mirshahi,
Commonwealth
University, Richmond, VA, J. Choudhury,
University,
Richmond, VA, A. Sanyal, Virginia Commonwealth University, Richmond,
VA
BACKGROUND
Many patients with HCV infection use cannabinoids.
Cannabinoid
receptor activation has both direct effects on
hepatocytes and T-cell function. These could affect the virologic response to
HCV treatment. The potential effects of cannabinoids on interferon-mediated
suppression of viral replication were not known.
SPECIFIC
AIMS
To define the effects of tetrahydrocannabinol (THC)
and methanandamide (MA) a CB receptor agonist on interferon-mediated
suppression of
HCV replication in hepatocytes.
METHODS
HCV virus replication was measured by negative strand
quantitative PCR in
a commercially available HCV replicon system (APATH).
The effects of increasing doses of MA (0-3 μM) and THC (0-1.5
μM) on HCV replication was first measured. Next, the effect of MA and
THC on α-interferon mediated suppression of HCV replication was
measured. The specificity of these effects were tested by pharmacologic
blockade of CB1 and CB2 receptors. Next, the effects of MA on
interferon-mediated increased on protein kinase R (PKR) and interferon response
factor
(IRF) mRNA (measured by qPCR), and protein (measured
by Western blot) were measured.
RESULTS
Compared to controls, MA and THC produced a 40-62%
decrease in HCV
replication. These effects were most marked after 12
hours of exposure. As expected, IFN (5000 IU/ml) produced a 90% inhibition of
HCV replication. MA as well as THC blocked the IFN-mediated inhibition of HCV
replication. These effects could be blocked by the CB1 receptor antagonist SR
141716 but not by a CB2 antagonist SR144528. MA and THC also blocked the
interferon-mediated increase in PKR and IRF mRNA and
protein.
CONCLUSIONS
THC and other CB1 receptor agonists can block the
IFN-mediated
suppression of HCV replication in a replicon system by
inhibiting IFN-induction of IRF and PKR. The clinical relevance of these data
should be assessed by correlation of cannabinoid use with virologic response to
IFN therapy.
Abstract ID: 67430
Category: JO6: HCV Therapy: Preclinical and Early Clinical Development
Itmn A and B, novel inhibitors of the HCV NS3/4 protease retain their potency against VX-950 and BILN-2016 resistant NS3/4 protease mutants and an IFN-a-2a resistant HCV replicon.
S.
Seiwert, InterMune, Brisbane, CA, S. W. Andrews, Array Biopharma, Boulder, CO,
H.
Yang, Gilead, Foster City, CA, H. Tan, InterMune, Brisbane, CA, B. Marifino,
InterMune,
Brisbane, CA, R. Radhakrishnan, InterMune, Brisbane, CA, E. Cheung,
InterMune,
Brisbane, CA, R. Rieger, Array Biopharma, Boulder, CO, Y. Jiang, Array
BioPharma,
Boulder, CO, A. Kennedy, Array BioPharma, Boulder, CO, S.
Wenglowsky,
Array BioPharma, Boulder, CO, M. Madduru, Array BioPharma, Boulder,
CO,
J. Bencsik, Array BioPharma, Boulder, CO, M. Liang, Array BioPharma, Boulder,
CA,
B. Woodward, Array BioPharma, Boulder, CO, K. Condroski, Array BioPharma,
Boulder,
CO, C. Lemieu, Array BioPharma, Boulder, CO, L. Pieti Opie, Array
BioPharma,
Boulder, CO, G. Hingorani, Array BioPharma, Boulder, CO, W. E.
Delaney,
Gilead, Foster City, CA, H. Yang, Gilead, Foster City, CA, A. Kaup,
University
of Texas Southwestern, Dallas, TX, M. Gale,
Southwestern,
Dallas, TX, J. Winkler, Array BioPharma, Boulder, CO, J. Josey, Array
BioPharma,
Boulder, CO, L. M. Blatt, InterMune,
Introduction
The standard of care (SoC) for chronic HCV infection,
PEG IFN
Α -2 and ribavirin, results in a sustained
virologic response in ~50% of patients, demonstrating a clear need for the
development of novel therapeutic approaches to treat this disease. We therefore
embarked on a
rational drug design campaign to produce inhibitors of
the HCV NS3/4 protease. Two lead compounds (ITMN A and ITMN B) emerged from our
discovery paradigm with EC50 potencies in biochemical and replicon assays of
<2 nM (genotype 1b), liver levels in multiple species predictive of
efficacious exposure in humans, and acceptable tolerability. Both of these
compounds have been designated preclinical candidates and
are currently undergoing INDenabling toxicological assessment. The activity of
both compounds on forms of the NS3/4 protease resistant to two other NS3/4
protease inhibitors, VX-950 and BILN-2016, was examined. Mutation of an alanine
at position 156 to a serine (A156S) is a dominant resistance
mutation to VX-950. ITMN A and ITMN B showed IC50s
against the A156S NS3/4 protease of 4.5 nM and 2.4 nM, respectively; roughly
1,000-fold more potent than VX-950. Similarly, D168V is a primary resistance
mutation to BILN-2061 and ITMN A and ITMN B showed EC50s
against the D168V NS3/4 protease of 10 nM and <2
nM, respectively.
In an effort to understand the molecular basis for the
~50% response rate to the SoC for chronic HCV, a subgenomic HCV replicon that
displayed more than a 28-fold reduced sensitivity to the antiviral effects of
IFN α-2a was evolved in vitro. This replicon partially evades interferon
responses by blocking the activation of interferon regulatory factor-3 (IRF-3)
and consequently the downstream expression of a repressor of viral translation
(ISG56). ITMN compounds retained their full potency against this interferon
“resistant” replicon and furthermore restored expression of ISG56.
Conclusion
Pre-clinical candidates ITMN A and ITMN B remain
highly active against NS3/4 mutants resistant to VX-950 and BILN-2061. Thus,
these compounds may have utility for the treatment of any naturally occurring-
or drug induced- HCV quasispecies containing A156S or D168V mutations in NS3/4.
In addition, the uncompromised activity of ITMN A and B on an
IFN a-2 insensitive HCV subgenomic replicon implies
that these two compounds, and perhaps NS3/4 protease inhibitors in general, may
disrupt viral processes that suppress response to interferon treatment.
Abstract ID: 65933
Category: JO6: HCV Therapy: Preclinical and Early Clinical Development
The immunomodulator AS101 inhibits HCV replication.
M. Shalita-Chesner, Rabin Medical Center, Petach
Tikva, Israel, R. Zemel, Rabin
Medical Center, Petach Tikva, Israel, M. Gal-Tanami,
Rabin Medical Center, Petach
Tikva, Israel, B. Sredni, Bar-Ilan University,
Ramat-Gan, Israel, L. Bachmatov, Rabin
Medical Center, Petach Tikva, Israel, I. Benhar,
Tel-Aviv University, Tel-Aviv, Israel,
R. Tur-Kaspa, Rabin Medical Center, Beilinson campus,
Petach-Tikva, Israel
Introduction
Current therapeutic options for hepatitis C virus
(HCV) infection are limited; therefore, alternative therapeutic approaches are
needed. Inhibition of HCV gene expression and replication as well as
immunotherapeutic concepts aimed at enhancing the cellular immune response
against HCV are being explored. One promising new strategy is the use of
immunomodulating agents in combination with IFN and/or ribavirin to treat HCV
patients. The nontoxic immunomodulator ammonium
trichloro (dioxoethylene-o,o') tellurate (AS101), first developed by us has
been shown to have beneficial effects in diverse preclinical and clinical
studies. Most of its activities have been primarily attributed to the direct
inhibition of the anti-inflammatory cytokine IL-10. This
immunomodulatoryproperty was found to be crucial for the clinical activities of
AS101, demonstrating the
protective effects of AS101 in viral-infected mice
models. AS101 have also inhibitory effect on different viral catalytic functions
such as inhibition of the HIV reverse transcriptase. Phase I clinical trials
with advanced cancer patients treated with AS101 showed increased production
and secretion of a variety of cytokines, leading to a clear dominance in TH1
responses with a concurrent decrease in the TH2 responses.
Aim
The aim of our study was to investigate the inhibitory
effect of the AS101 on the catalytic activity of HCV NS3 serine protease in
order to evaluate its potential as an HCV therapeutic agent. The effect of AS101
on HCV serine protease activity was analyzed in vitro using the fluorometric
assay developed in our laboratory. This effect was further confirmed by our
novel high-throughput in-vivo genetic screen. This screen is based on
the concerted co-expression of a reporter gene and
recombinant NS3 in E. coli. The effect of AS101 on HCV in-vivo was determined
using a cell-based selectable subgenomic HCV RNA Huh-7 replicon system. In this
system HCV RNA replication is monitored in intact cells by the enzymatic assay
of placental alkaline phosphatase (SEAP) activity secreted.
Results
Our study demonstrates that AS101 specifically
inhibits NS3 serine protease
catalysis in a dose-dependent manner in the assay system
examined. It reaches 70.3 ± 9.5% inhibition at AS101 concentration of 10 mg/ml.
Moreover, AS101 specifically inhibited HCV replication as determined in Huh-7
replicon system.
Conclusion
Our results suggest that HCV NS3 serine protease is a novel target for AS101 action and of the inhibitory effect of AS101 on HCV replication involve repression of HCV NS3 serine protease.
Abstract ID: 67536
Category: JO6: HCV Therapy: Preclinical and Early Clinical Development
Characterization of in vitro selected Con 1 subgenomic replicons resistant to 2’-Cmethyl-cytidine, a potent inhibitor of genotype 1a and 1b HCV polymerases.
S. Rajyaguru, Roche Palo Alto LLC, Palo Alto, CA,
S. Le Pogam, Roche Palo Alto
LLC, Palo Alto , CA, V. Leveque, Roche Palo LLC,
Palo Alto, CA, H. Kang, Roche
Palo Alto LLC, Palo Alto, CA, H. Ma, Roche Palo
Alto LLC, Palo Alto, CA, S. Jiang,
Roche Palo Alto LLC, Palo Alto, CA, K. Klumpp,
Roche Palo Alto LLC, Palo Alto, CA,
J. Symons, Roche Palo Alto LLC, Palo Alto, CA, N.
Cammack, Roche Palo Alto LLC,
Palo Alto, CA, I. Najera, Roche Palo Alto LLC,
Palo Alto, CA
Introduction
Characterization of in vitro selected Con 1 subgenomic
replicons resistant to 2’-Cmethyl- cytidine, a potent inhibitor of genotype 1a
and 1b HCV polymerases. The HCV polymerase is an attractive target for the
development of new and HCV specific antiviral compounds. 2’-C-methyl-cytidine
is a potent inhibitor of both genotype 1b (GT-1b) and GT-1a-polymerase driven
replicon replication (IC50 values of 1.0 μ M). The corresponding
5’-triphosphate derivative is a potent and selective inhibitor of native HCV
replicase isolated from replicon cells (IC50 value of 0.22
Μ M), and of the recombinant GT-1b and GT-1a HCV
polymerase mediated RNA synthesis, (IC50 values of 0.167 μ M for BK NS5B
and 0.24 μ M for H77 NS5B). Previous studies have shown that the replicon
system allows the selection of replicon variants resistant to HCV inhibitors.
Methods
In this study, we used the HCV subgenomic replicon
system to select and
characterize HCV variants with reduced susceptibility
(>20-fold compared to wild type) to 2’-C-methyl-cytidine.
Results
Characterization of resistant replicons by sequence
analysis confirmed the presence of amino acid substitution(s) in the NS5B
coding region. Site directed mutant transient replicons confirmed the role of
the observed mutation(s) in the resistant phenotype. The HCV replicase complex
isolated from cells containing mutant replicons as well as the mutant
recombinant polymerase-mediated RNA synthesis further confirmed the observed
reduced susceptibility to the corresponding 5’-triphosphate derivative. Cross
resistance was observed with 2’-C-methyl-adenosine. However, no
cross resistance was observed with the nucleoside
analog R1479 or with the non nucleoside, benzothiadiazine- or thiophene
carboxylic acid-derivative HCV polymerase inhibitors.
Conclusion
This data will allow the optimization of new
polymerase inhibitors and their use in combination therapy.
Abstract ID: 67538
Category: JO6: HCV Therapy: Preclinical and Early Clinical Development
An á-glucosidase inhibitor approved for use in type II diabetes has antiviral activity and displays synergy with IFN alfacon-1 in a surrogate system for HCV.
H.
Tan, InterMune, Brisbane, CA, S. Seiwert, InterMune, Brisbane, CA, L. M. Blatt,
InterMune,